PROJECT SUMMARY Ischemic complications including myocardial infarction are the leading causes of mortality among patients with complement-mediated diseases including transplant arteriosclerosis (TA) and autoimmune connective tissue disease. Complement are a set of nine circulating immune proteins involved in host defense that, when activated under disease conditions, cause endothelial cell (EC) dysfunction. Upon activation by Ab, complement proteins self-assemble into transmembranous, pore-like structures called membrane attack complexes (MAC). MAC insert into EC surfaces where they initiate inflammatory signaling without causing cell death. Understanding the inflammatory signals driven by MAC to EC may lead to new therapies blocking the ischemic sequelae of complement-mediated diseases. To study these processes, I used panel reactive antibody (PRA) sera from alloantigen-sensitized transplant candidates to induce Ab-mediated MAC assembly on human EC in vitro and in vivo. In this process, I discovered a novel MAC effector pathway, non-canonical NF-?B, characterized by rapid and dramatically increased NF-?B inducing kinase (NIK). NIK induced inflammatory genes, enhanced the ability of EC to activate CD4+ T cells, and was implicated in TA, a vaso-occlusive condition occurring as a complication of heart transplantation, in 2 humanized mouse models. To understand how MAC could induce NIK, I performed a genome-wide siRNA screen and uncovered a novel, endosome-based mechanism by which MAC stabilized NIK. MAC was rapidly internalized and transferred to Rab5+ vesicles to form a MAC+Rab5+ compartment. In a Rab5-dependent manner, the MAC+Rab5+ compartment sequentially recruited activated Akt (pAkt) and NIK. Here, Rab5 activity was required to recruit activated Akt to MAC+Rab5+ endosome to induce NIK stabilization. To define mechanism(s) by which Rab5 could recruit pAkt, I performed proteomic analyses of FACS-sorted MAC+Rab5+ endosomes and defined a Rab5-ZFYVE21-SMURF2 signaling axis causing NIK stabilization and EC dysfunction. I hypothesize that MAC activates a Rab5-ZFYVE21-SMURF2 axis that elicits EC dysfunction by stabilizing NIK. In Aim 1, I will define three mechanism(s) regulating the Rab5-ZFYVE21-SMURF2 axis. I will examine 1) how lipid-associated kinases and phosphatases mediate lipid remodeling of MAC+Rab5+ endosomes to stabilize NIK, 2) how MAPK(s) regulate Rab5 activity, and 3) how pAkt is recruited to MAC+Rab5+ endosomes. In Aim2 I will examine the relevance of select molecules regulating Rab5-ZFYVE21- SMURF2 signaling in patient tissues, in humanized models of EC signaling in vivo, and in a humanized mouse model of TA. The long-term aims of this proposal are to better understand MAC-induced inflammatory signals. By doing this, druggable targets ameliorating MAC-induced ischemic complications may emerge.